Therapeutic il-13 polypeptides

ABSTRACT

Variant IL-13 polypeptides are provided, which are engineered to have one or more of the following properties: (a) altered affinity for IL-13Rα2, relative to the native human IL-13 protein; (b) altered affinity for IL-13Rα1 relative to the native human IL-13 protein; (c) a disruption in the binding site for IL-4Rα relative to the native human IL-13 protein.

CROSS REFERENCE

This application claims benefit and is a Divisional of application Ser.No. 15/353,273 filed Nov. 16, 2016, which is a Divisional of applicationSer. No. 14/373,498 filed Jul. 21, 2014, which is a 371 application andclaims the benefit of PCT Application No. PCT/US2013/023194, filed Jan.25, 2013, which claims benefit of U.S. Provisional Patent ApplicationNo. 61/591,781, filed Jan. 27, 2012, which applications are incorporatedherein by reference in their entirety.

BACKGROUND

Interleukin-13 (IL-13) is a cytokine secreted by T lymphocytes and mastcells, which shares several biological activities with IL-4, as amediator of allergic inflammation and disease. IL-13 is involved in theallergic response via its actions on epithelial and smooth muscle cells.IL-13 induces many features of allergic lung disease, including airwayhyperresponsiveness, goblet cell metaplasia and mucus hypersecretion,which all contribute to airway obstruction. IL-13 also induces secretionof chemokines that are required for recruitment of allergic effectorcells to the lung. Many polymorphisms in the IL-13 gene have been shownto confer an enhanced risk of atopic respiratory diseases such asasthma.

The art has shown that neutralization of endogenously released IL-13with a soluble form of IL-13Rα2, which binds IL-13 but not IL-4, duringantigen exposure largely inhibited the characteristics of asthma inmurine asthma models. In addition, antigen challenge of IL-13-deficientmice failed to elicit airway hyperresponsiveness and mucus productiondespite the continued presence of IL-4 and IL-5 and airway inflammation.Its importance as an effector molecule in asthma was further evidencedby the finding that acute administration of IL-13 itself was sufficientto recapitulate many features of airway responses characterized inasthma, such as eosinophilic inflammation, mucus hyperproduction andairway hyperresponsiveness in mice. It has been suggested that IL-13,independent from other Th2 cytokines, is necessary and sufficient toinduce key features of allergic inflammation at an effector phase.

Various biologicals have been tested for treatment of asthma, includinghumanized monoclonal antibodies (MoAbs) against IL-5 and IL-13, and arecombinant human soluble IL-4 receptor (sIL-4R). For example, toevaluate the biologic and clinical relevance of interleukin-13 inpatients with uncontrolled asthma, lebrikizumab, an IgG4 humanizedmonoclonal antibody that specifically binds to interleukin-13 andinhibits its function was administered. Treatment with lebrikizumab wasassociated with a significant improvement in prebronchodilator FEV₁, theprimary outcome.

An important factor in IL-13 biology is the nature of its receptorinteractions. Its diverse functions are mediated by a complex receptorsystem including IL-4 receptor α (IL-4Rα; CD124) and two other cognatecell surface proteins, IL-13Rα1 (CD213a1) and IL-13Rα2 (CD213a2).IL-13Rα1 forms a heterodimer with IL-4Rα that is a signaling IL-13receptor. In contrast, IL-13Rα2 has been thought to be a decoy receptordue to its short cytoplasmic tail. IL-13Rα2 exists on the cell membrane,intracellularly, and in soluble form. Recent reports revealed thatmembrane IL-13Rα2 may have some signaling capabilities, and solubleIL-13Rα2 may be an endogenous modulator for IL-13 responses. IL-13Rα2has an extremely high affinity for IL-13, and can actually out-competeantibodies for IL-13 binding. The other receptor, IL-13Rα1, has a muchlower affinity, but is associated with signaling events mediated byIL-4Rα. It induces its effects through a multi-subunit receptor thatincludes the alpha chain of the IL-4 receptor (IL-4Rα) and IL-13Rα1.Most of the biological effects of IL-13, like those of IL-4, are linkedto a single transcription factor, signal transducer and activator oftranscription 6 (STATE).

Biologicals that provide for selective alteration of IL-13 activity areof interest for a number of therapeutic purposes, including thetreatment of asthma and atopy, and certain cancers. The presentinvention addresses this issue.

SUMMARY OF THE INVENTION

IL-13 polypeptides and analogs thereof are provided, which polypeptidesprovide for selective binding to IL-13 receptors. Certain of thepolypeptides of the invention are highly selective antagonists of nativeIL-13 activity. Other peptides are selective agonists of native IL-13activity.

The IL-13 polypeptides of the invention are engineered to have one ormore of the following properties: (a) altered affinity for IL-13Rα2,relative to the native human IL-13 protein. Such peptides may havedecreased affinity, which polypeptides may be designated herein as(R2^(lo)); or alternatively may have increased affinity, whichpolypeptides may be designated herein as (R2⁺); (b) altered affinity forIL-13Rα1 relative to the native human IL-13 protein. Such peptides mayhave decreased affinity, which polypeptides may be designated herein as(R1^(lo)); or alternatively may have increased affinity, whichpolypeptides may be designated herein as (R1^(hi)); (c) a disruption inthe binding site for IL-4Rα relative to the native human IL-13 protein,which polypeptides may be referred to herein as (4R^(null)).

In some embodiments, amino acid modifications are made at one or more ofthe amino acids within the set of contact residues that interact withIL-13Rα1, IL-13Rα2 or IL-4Rα1, which residues include, withoutlimitation, L10, R11, I14, E12, V18, R65, R86, D87, T88, K89, L101,K104, K105, F107, and R108 (for reference purposes the sequence ofwild-type human IL-13 is provided herein as SEQ ID NO:1, to which thenumbering of amino acids will refer). In other embodiments, modifiedresidues are at two or more, three or more, four or more, five or more,and not more than 14 amino acids within the combined set of contactresidues defined above.

The IL-13 interface that contacts IL-13Rα1 and IL-13Rα2 is the same, andthus there can be overlap in the altered residues that control affinityfor these two receptors. In some embodiments one or more of the nativeamino acid residues L10, R11, I14, V18, R86, D87, T88, K89, L101, K104,K105, F107, and R108 is substituted, and provides for an alteredaffinity for one or both of IL-13Rα1 and IL-13Rα2.

In some embodiments an IL-13 peptide of the invention comprises one ormore of the amino acids substitutions: (1) L10F; L101; L10V; L10A; L10D;L10T; L10H; (2) R11S; R11N; R11H; R11L; R11I; (3) I14L; I14F; I14V;I14M; (4) V18L; V18F; V18I; (5) E12A; (6) R65D; (7) R86K; R86T; R86M;(8) D87E; D87K; D87R; D87G; D87S; (9) T881; T88K; T88R; (10) K89R; K89T;K89M; (11) L101F; L101I; L101Y; L101H; L101N; (12) K104R; K104T; K104M;(13) K105T; K105A; K105R; K105E; (14) F107L; F1071; F107V; F107M; and(15) R108K; R108T; R108M, which substitutions cause an altered affinityfor one or both of IL-13Rα1 and IL-13Rα2. In other embodiments, modifiedresidues are at two or more, three or more, four or more, five or more,and not more than 14 amino acids within the combined set of contactresidues defined above.

In some embodiments an IL-13 polypeptide of the invention is an IL-13agonist. Agonists have increased affinity for one or both of IL-13Rα1and IL-13Rα2; and may have the properties of being R1^(hi)R2^(lo) or maybe R1^(lo)R2⁺. Such variants find use in various methods, includingwithout limitation targeting of cancer cells, induction of apoptosis inIL-13 receptor positive cells; and the like.

In other embodiments an IL-13 polypeptide of the invention is anantagonist of IL-13 signaling. Antagonists may be 4R^(null), and inparticular may be R1^(hi)R2^(lo)4R^(null), which antagonists find use ininhibiting the signaling of endogenous IL-13, e.g. for use in treatmentof asthma, atopic disease, and other conditions in which there isundesirable IL-13 activity. Alteration of the native amino acid residuesE12 and R65 can be sufficient to ablate binding to IL-4Rα, for examplethe amino acid substitutions E12A and R65D.

The invention also includes pharmaceutical formulations of IL-13agonists or antagonists, in combination with a pharmaceuticallyacceptable excipient. Such formulations may be provided as a unit dose,e.g. a dose effective in inhibition of endogenous IL-13 signaling in anindividual, for targeting to IL-13R⁺ cells, etc. Pharmaceuticalformulations also include lyophilized or other preparations of thepolypeptides of the invention, which may be reconstituted for use.

In some embodiments, methods for therapeutic treatment of atopicdisease, including asthma, are provided. Such methods compriseadministering to an individual in need thereof an effective dose of anIL-13 antagonist of the invention, wherein the antagonist reduces theundesirable effects of endogenous IL-13 signaling, which may include,for example, eosinophilic inflammation, mucus hyperproduction and airwayhyperresponsiveness. In such methods the administration of the IL-13antagonist may be localized, e.g. to the lungs, or systemic. In someembodiments an aerosol formulation to the lungs is the mode ofadministration.

In some embodiments, methods are provided for targeting tumor cellsexpressing IL-13Rα2, which tumors include, without limitation,glioblastoma cells. In such methods the tumor cells are contacted withan R1^(lo)R2^(hi) agonist, which selectively targets the IL-13Rα2receptor. The agonist may be conjugated to a markers for imaging, e.g. afluorophore, radiochemical, enzyme marker, etc.; and may be conjugatedto a therapeutic agent, e.g. a cytotoxin, a radioactive element, and thelike.

In some embodiments, methods are provided for increasing signalingthrough IL-13Rα 1. In such methods a cell of interest is contacted withan effective dose of an R1^(hi)R2^(lo) agonist, which selectively bindsto IL-13Rα1. The dose may be effective to induce apoptosis in the cellof interest. The contacting step may be in vivo, e.g. for therapeuticpurposes; or in vitro, e.g. for screening and research purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Crystal structure of the IL-13 ternary ectodomain complex. SiteII and site III interfaces are indicated with a red circle. Left panelsshow a zoom in on the interfaces where representative positions mutatedin the site II (helix A and D) and site III (C-D loop) are highlightedin orange. IL-13 is in orange, IL-13Rα1 is in purple and IL-4Rα1 is incyan.

FIG. 2A-2B. Comparative analysis of the IL-13Rα1- and theIL-13Rα2-selective IL-13 variants (FIG. 2A) Human IL-13 and IL-13Rα1 andIL-13Rα2 selective variants sequences are given for the indicatedresidue numbers. Kinetic and affinity parameters were determined bysurface plasmon resonance. (FIG. 2B) Histogram representation of thenormalized K_(D) binding affinity values for IL-13Rα1 (purple) andIL-13Rα2 (orange) selective variants. IL-13 wt K_(D) value wasnormalized to one and the rest of the values were changed accordingly.

FIG. 3A-3B. Signaling activation induced by the IL-13Rα1- and theIL-13Rα2-selective IL-13 variants. (FIG. 3A) The IL-13 responsive cellline A549 was stimulated with doses ranging from 500 nM to 5E-06 nM ofthe different IL-13 variants for fifteen minutes. Cells were then fixedand permeabilized with 100% cold methanol and stained with antibodyagainst phosphorylated Stat6. The percentage of the MFI value was usedto plot the data. (FIG. 3B) A549 cells were stimulated with 500 nM ofthe IL-13 variants for the indicated times, fixed, permeabilized andstained with phospho-specific Stat antibodies as indicated above and theratio P-Stat6/P-Stat3 was plotted against time.

FIG. 4A-4B. Functional outcomes induced by the IL-13Rα1- and theIL-13Rα2-selective IL-13 variants. (FIG. 4A) Human monocytes werepurified from peripheral blood mononuclear cells and cultured with 50ng/ml GM-CSF alone or with the indicated doses of the different IL-13variants Cells were analyzed on day 6 with mAbs against HLA-DR, CD86,CD209. Data (mean and SEM) are from 3 donors. (FIG. 4B) TF-1 cells wereseeded in a p96 well plate (100.000 cells/well) and stimulated for fivedays with the indicated doses of the IL-13 variants. Cells were thenwashed twice with cold PBS and fixed with 4% PFA. The number of cells ineach well was determined by flow cytometry. The experiment was repeatedthree times and the means and SEMs were plotted versus the concentrationof cytokine used.

FIG. 5A-5B. Crystal structure of the IL-13 ternary ectodomain complex.(FIG. 5A) Site I is highlighted with a red circle. Left panel show acloser look of the site I interface where amino acids mutated in the Chelix are colored in orange. IL-13 is in orange, IL-13Rα1 is in purpleand IL-4Rα1 is in cyan. (FIG. 5B) Sequences for human IL-13 and IL-13dnare given for the indicated residue numbers. Kinetic and affinityparameters were determined by surface plasmon resonance

FIG. 6A-6C. Analysis of the IL-13dn efficacy in vitro. (FIG. 6A) TheIL-13 responsive cell line THP-1 was stimulated with different doses ofIL-13 (pink), IL-13dn (orange) and IL-13 in the presence of 50 nM ofIL-13dn (green) for fifteen minutes. Cells were fixed and permeabilizedwith 100% cold methanol and analyzed for Stat6 phosphorylation usingphospho-specific antibodies coupled to Alexa-488 fluorochrome. (FIG. 6B)The macrophage cell line Raw264 stably expressing a Stat6 luciferasereporter was seed in a p96 well plate (100.000 cells/plate) and thenstimulated with 10 ng/ml of mouse IL-13 in the presence of the indicateddoses of IL-13dn for 48 hr. Cells were lysed with 30 μl of lysis buffer(Promega) and the luciferase activity was measured for 1 sec. (FIG. 6C)Human monocytes were purified from peripheral blood mononuclear cellsand cultured with 50 ng/ml GM-CSF alone or with 20 ngr/ml of IL-13 wt inthe presence of 2 μg/ml of isotype antibody control, anti-IL4Rα1antibody or IL-13dn. Cells were analyzed on day 6 with mAbs againstCD14, CD86, CD209. Data (mean and SEM) are from 3 donors.

FIG. 7A-7B. Analysis of the IL-13dn efficacy in vivo. (FIG. 7A)Schematic flowchart where the doses and times used to test the efficacyof IL-13dn in vivo are indicated. (FIG. 7B) qPCR analysis of theexpression levels of the Th2 inflammation markers (Muc5ac, Periostin,Arg¹, CHIA, YM1, Fizz1) induced by mouse IL-13 in the presence of theindicated dose of IL-13dn.

DEFINITIONS

In the description that follows, a number of terms conventionally usedin the field of cell culture are utilized. In order to provide a clearand consistent understanding of the specification and claims, and thescope to be given to such terms, the following definitions are provided.

The terms “inhibitors,” “antagonists” refer to an agent that reduces theeffective biological activity of IL-13 present in the system, e.g. ananimal, a tissue, an in vitro culture system, etc., for exampleendogenous IL-13 in an individual, usually by interfering with theinteraction between IL-13 and one or more of its receptors. For example,an antagonist of the invention may bind tightly to the IL-13Rα1receptor, but have low affinity to IL-13Rα2, so that it is not “trapped”by that receptor. Antagonists may also have ablated binding to IL-4Rα,to prevent signaling through that receptor. For development purposes thebinding may be performed under experimental conditions, e.g. usingisolated proteins as binding partners, using portions of proteins asbinding partners, using yeast display of proteins or portions ofproteins as binding partners, and the like.

Altered Affinity for IL-13Rα2, Relative to the Native Human IL-13Protein.

The human interleukin 13 receptor, alpha 2 (IL13RA2) may be referencedwith the genetic sequence of Genbank accession number NM_000640. Thepredicted 380-amino acid protein contains a putative signal sequence, anextracellular region with a fibronectin-like domain and typical cytokinereceptor motifs, a transmembrane domain, and a short intracellular tail.Amino acid substitutions that provide for altered Rα2 affinity includewithout limitation (1) L10H; L10A; (2) R11L; (4) V18I; (7) R86M; R86K;R86T; (8) D87K; D87G; (9) T88S; T88K; (10) K89R; (11) L101N; (12) K104R;(13) K105A; K105E; (14) R108K.

IL13 binds with high affinity to IL13RA1, which inducesheterodimerization with IL4R to form a complex, or alternatively, IL13may bind with even greater affinity to IL13RA2, which fails to induce asignal, indicating that it acts as a decoy receptor. The C-terminaltails of the IL4 and IL13 receptor subunits interact with tyrosinekinases of the Janus kinase family (e.g., JAK1), leading to interactionwith STATE, which binds to consensus sequences in the promoters of IL4-and IL13-regulated genes.

Affinity for IL-13Rα2 to wild-type IL-13 is extremely high, andtherefore only modest increases in affinity will be found inpolypeptides of the invention, for example equivalent, 2-fold increase,3-fold increase, 5-fold increase of kinetic K_(D). With reference toFIG. 2A, peptide C11 (SEQ ID NO:18) and D7 (SEQ ID NO:20) showequivalent or slightly increased binding to IL-13Rα2, and have the setof amino acid substitutions [L10H, R86T, D87G, T88R, R108K] and [L10A,V18F, R86K, D87K, K89R, L101I, K104R, R108K], respectively.

Decreased affinity for IL-13Rα2 is of interest for various purposes,including in particular the design of antagonists. Polypeptidesdesignated as R2^(lo) may have a kinetic K_(D) that is about 5-fold,about 10-fold, about 10²-fold, about 5×10²-fold, about 10³-fold lowerthan a native IL-13 polypeptide. Included in the class is thepolypeptide DN (SEQ ID NO:2) [L10V, V18I, D87S, D88S, L101F, K104R,K105T, and in addition the IL-4R binding residues E12A, R65D].

Altered Affinity for IL-13Rα1 Relative to the Native Human IL-13Protein.

The human interleukin 13 receptor, alpha 1 (IL13RA1) may be referencedwith the genetic sequence of Genbank accession number NM_001560. It is aprotein of 424 amino acid residues, containing a putative signalsequence and transmembrane domain, which is a low-affinity receptor.Amino acid substitutions that provide for altered Rα1 affinity includewithout limitation (1) L101; L10V; (4) V18I; (7) R86K; R86M; (8) D87G;D87S; (9) T88S; (10) K89R; K89M; (11) L101H; L101Y; (12) K104R; (13)K105A; K105T.

Decreases in affinity may be modest, for example equivalent, 2-folddecrease, 3-fold decrease, 5-fold decrease of kinetic K_(D). Decrease inaffinity can also be greater than about 10-fold, greater than about10²-fold, greater than about 10³-fold or more. For example, withreference to FIG. 2, polypeptide B4 (SEQ ID NO:9) provides a decreasedaffinity to IL-13Rα1, and has the set of amino acid substitutions [R11S,V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T]. Polypeptide C4provides a decreased affinity to IL-13Rα1, and has the set of amino acidsubstitutions [L10V, K89R, L101N, K105E, R108T].

Increased affinity for IL-13α1 is of interest for various embodiments ofthe invention, in particular when coupled with decreased or equivalentaffinity to IL-13Rα2, for example in DN, SEQ ID NO:2. Polypeptidesdesignated as R1^(hi) may have a kinetic K_(D) that is about 5-fold,about 10-fold, about 10²-fold, about 5×10²-fold, about 10³-fold higherthan a native IL-13 polypeptide. Polypeptides of interest having thisproperty include A11 (SEQ ID NO:4); B2 (SEQ ID NO:8); A5 (SEQ ID NO:5).Sets of amino acid substitutions for A11, B2, and A5, respectively, areas follows: [L10V, V18I, D87S, T88S, L101F, K104R, K105T]; [V18I, R86T,D87G, T88S, L101Y, K104R, K105A]; and [R11I, V18I, R86K, D87G, T88S,L101H, K104R, K105A, F107M].

Disruption in the Binding Site for IL-4Rα Relative to the Native HumanIL-13 Protein.

The human interleukin 4, alpha 1 (IL4R) may be referenced with thegenetic sequence of Genbank accession number NM_000418. Membrane-boundIL4R is coded by exons 3 to 7 (extracellular domain), exon 9(transmembrane domain), and exons 10 to 12 (intracellular domain).Alternative splicing leads to the production of a soluble form of IL4R,which is coded by exons 3 to 8 and lacks the exons for the transmembraneand intracellular regions.

Amino acid substitutions at residues E12 and R65 (relative to SEQ IDNO:1) are sufficiently to substantially ablate binding to the IL-4R,which variants may be references as 4R^(null). In some embodiments theamino acid substitutions are E12A and R65D, in other embodiments theamino acids are conservative variants thereof, e.g. E125, E12G, etc.;R65E, and the like.

The binding properties of a binding agent may be measured by any method,e.g., one of the following methods: BIACORE™ analysis, Enzyme LinkedImmunosorbent Assay (ELISA), x-ray crystallography, sequence analysisand scanning mutagenesis. The ability of a protein to neutralize and/orinhibit one or more IL-13-associated activities may be measured by thefollowing methods: assays for measuring the proliferation of an IL-13dependent cell line, e.g. TFI; assays for measuring the expression ofIL-13-mediated polypeptides, e.g., flow cytometric analysis of theexpression of CD23; assays evaluating the activity of downstreamsignaling molecules, e.g., STATE; assays evaluating production oftenascin; assays testing the efficiency of an described herein toprevent asthma in a relevant animal model, e.g., the cynomolgus monkey,and other assays. An IL-13 polypeptide can have a statisticallysignificant effect in one or more of these assays. Exemplary assays forbinding properties include the following.

The binding interaction of a IL-13 polypeptide and a target (e.g.,receptor) can be analyzed using surface plasmon resonance (SPR). SPR orBiomolecular Interaction Analysis (BIA) detects biospecific interactionsin real time, without labeling any of the interactants. Changes in themass at the binding surface (indicative of a binding event) of the BIAchip result in alterations of the refractive index of light near thesurface. The changes in the refractivity generate a detectable signal,which are measured as an indication of real-time reactions betweenbiological molecules. Methods for using SPR are described, for example,in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons SpringerVerlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szaboet al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resourcesprovide by BIAcore International AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitativemeasure of the equilibrium dissociation constant (Kd), and kineticparameters, including Kon and Koff, for the binding of a molecule to atarget. Such data can be used to compare different molecules.Information from SPR can also be used to develop structure-activityrelationships (SAR). For example, the kinetic and equilibrium bindingparameters of different molecule can be evaluated. Variant amino acidsat given positions can be identified that correlate with particularbinding parameters, e.g., high affinity and slow Koff. This informationcan be combined with structural modeling (e.g., using homology modeling,energy minimization, or structure determination by x-ray crystallographyor NMR). As a result, an understanding of the physical interactionbetween the protein and its target can be formulated and used to guideother design processes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it is derived. The term refers topreparations where the isolated protein is sufficiently pure to beadministered as a therapeutic composition, or at least 70% to 80% (w/w)pure, more preferably, at least 80%-90% (w/w) pure, even morepreferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%,98%, 99%, or 100% (w/w) pure. A “separated” compound refers to acompound that is removed from at least 90% of at least one component ofa sample from which the compound was obtained. Any compound describedherein can be provided as an isolated or separated compound.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having disease. Subjects may be human, but also includeother mammals, particularly those mammals useful as laboratory modelsfor human disease, e.g. mouse, rat, etc.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas disease cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc. The term “biological sample” encompasses a clinicalsample, and also includes tissue obtained by surgical resection, tissueobtained by biopsy, cells in culture, cell supernatants, cell lysates,tissue samples, organs, bone marrow, blood, plasma, serum, and the like.A “biological sample” includes a sample obtained from a patient'sdisease cell, e.g., a sample comprising polynucleotides and/orpolypeptides that is obtained from a patient's disease cell (e.g., acell lysate or other cell extract comprising polynucleotides and/orpolypeptides); and a sample comprising disease cells from a patient. Abiological sample comprising a disease cell from a patient can alsoinclude non-diseased cells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of death or progression, including recurrence, spread, anddrug resistance. The term “prediction” is used herein to refer to theact of foretelling or estimating, based on observation, experience, orscientific reasoning. In one example, a physician may predict thelikelihood that a patient will survive.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of effecting a partial or complete cure fora disease and/or symptoms of the disease. “Treatment,” as used herein,may include treatment of an atopic disorder or tumor in a mammal,particularly in a human, and includes: (a) preventing the disease or asymptom of a disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it(e.g., including diseases that may be associated with or caused by aprimary disease; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, i.e., causing regression ofthe disease.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of a disease, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withdisease or other diseases. The term “therapeutic effect” refers to thereduction, elimination, or prevention of the disease, symptoms of thedisease, or side effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

“Concomitant administration” of a known disease therapeutic drug with apharmaceutical composition of the present invention means administrationof the drug and IL-13 polypeptide at such time that both the known drugand the composition of the present invention will have a therapeuticeffect. Such concomitant administration may involve concurrent (i.e. atthe same time), prior, or subsequent administration of the drug withrespect to the administration of a compound of the invention. A personof ordinary skill in the art would have no difficulty determining theappropriate timing, sequence and dosages of administration forparticular drugs and compositions of the present invention.

Allergy, or Atopy is an increased tendency to IgE-based sensitivityresulting in production of specific IgE antibody to an immunogen,particularly to common environmental allergens such as insect venom,house dust mite, pollens, molds or animal danders. Allergic responsesare antigen specific. The immune response to the antigen is furthercharacterized by the over-production of Th2-type cytokines, e.g. IL-4,IL-5 and IL-10, by the responding T cells. The sensitization occurs ingenetically predisposed people after exposure to low concentrations ofallergen; cigarette smoke and viral infections may assist in thesensitization process.

Included in the group of patients suffering from atopy are those withasthma associated allergies. About 40% of the population is atopic, andabout half of this group develop clinical disease ranging from trivialrhinitis to life-threatening asthma. After sensitization, continuingexposure to allergens leads to a significant increase in the prevalenceof asthma. Ninety percent of children and 80% of adults with asthma areatopic. Once sensitization has occurred, re-exposure to allergen is arisk factor for exacerbations of asthma. Effective management ofallergic asthma includes pharmacological therapy and allergen avoidance.The specific physiological effects of asthma associated allergiesinclude airway inflammation, eosinophilia and mucus production, andantigen-specific IgE and IL-4 production.

Asthma is a respiratory disorder characterized by airway hyperreactivityand inflammation, and is associated with high serum IgE andoverproduction of interleukin (IL)-4, IL-5 and IL-13 byallergen-specific Th2 cells.

Cancers of interest include carcinomas, e.g. colon, prostate, breast,melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma, etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia,malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-celllymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoidhyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, etc.; sarcomas, melanomas, adenomas; benign lesions such aspapillomas, and the like.

The present methods are applicable to brain tumors, particularlyglioblastoma. In general, the goals of brain tumor treatments are toremove as many tumor cells as possible, e.g. with surgery, kill as manyof the cells left behind after surgery as possible with radiation and/orchemotherapy, and put remaining tumor cells into a nondividing,quiescent state for as long as possible with radiation and chemotherapy.Careful imaging surveillance is a crucial part of medical care, becausetumor regrowth requires alteration of current treatment, or, forpatients in the observation phase, restarting treatment.

Brain tumors are classified according to the kind of cell from which thetumor seems to originate. Diffuse, fibrillary astrocytomas are the mostcommon type of primary brain tumor in adults. These tumors are dividedhistopathologically into three grades of malignancy: World HealthOrganization (WHO) grade II astrocytoma, WHO grade III anaplasticastrocytoma and WHO grade IV glioblastoma multiforme (GBM). WHO grade IIastrocytomas are the most indolent of the diffuse astrocytoma spectrum.Astrocytomas display a remarkable tendency to infiltrate the surroundingbrain, confounding therapeutic attempts at local control. These invasiveabilities are often apparent in low-grade as well as high-grade tumors.

Glioblastoma multiforme is the most malignant stage of astrocytoma, withsurvival times of less than 2 years for most patients. Histologically,these tumors are characterized by dense cellularity, high proliferationindices, endothelial proliferation and focal necrosis. The highlyproliferative nature of these lesions likely results from multiplemitogenic effects. One of the hallmarks of GBM is endothelialproliferation. A host of angiogenic growth factors and their receptorsare found in GBMs.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Variant IL-13 polypeptides and analogs thereof are provided. The agentshave altered affinity for one or more receptors selected from IL-13Rα1,IL-13Rα2 and IL-4R, as described above in detail. Polypeptides havingincreased affinity for at least one receptor may find use as agonistsfor biological activity, and for targeting cells expressing the at leastone receptor, e.g. for delivery of therapeutic agents, labelingpurposes, and the like. Polypeptides that compete with the native IL-13protein for binding, and which preferably also are ablated for signalingthrough the IL-4R find use as antagonists, for example in the treatmentof conditions in which there is undesirable IL-13 signaling, such asatopy and asthma.

According to the present invention, amino acid modifications include anynaturally occurring or man-made amino acid modifications known or laterdiscovered in the field. In some embodiments, amino acid modificationsinclude any naturally occurring mutation, e.g., substitution, deletion,addition, insertion, etc. In some other embodiments, amino acidmodifications include replacing existing amino acid with another aminoacid, e.g., a conservative equivalent thereof. In yet some otherembodiments, amino acid modifications include replacing one or moreexisting amino acids with non-natural amino acids or inserting one ormore non-natural amino acids. In still some other embodiments, aminoacid modifications include at least 1, 2, 3, 4, 5, 6, 8, 10, 12 or 14amino acid mutations or changes. In some exemplary embodiments, one ormore amino acid modifications can be used to alter properties of theIL-13 polypeptide, e.g., affecting the binding activity and/orspecificity, etc. Techniques for in vitro mutagenesis of cloned genesare known in the art and described in the Examples herein.

IL-13 polypeptides of the invention may be at least about 50 amino acidsin length, at least about 75, at least about 100, at least about 110, atleast about 115 amino acids in length, up to the full-length of thewild-type protein at the transmembrane domain, i.e. about 116 aminoacids in length, and are optionally fused to a heterologous polypeptideor conjugated to a carrier molecule such as PEG, etc. Exemplarypolypeptide sequences are provided in SEQ ID NO:2-SEQ ID NO:20

Sets of modifications may include the following specific changes: (1)L10H; L10A; (2) R11L; (4) V18I; (7) R86M; R86K; R86T; (8) D87K; D87G;(9) T88S; T88K; (10) K89R; (11) L101N; (12) K104R; (13) K105A; K105E;(14) R108K.

Specific sets of modifications may include, without limitation:

-   -   [L10H, R86T, D87G, T88R, R108K]    -   [L10A, V18F, R86K, D87K, K89R, L101I, K104R, R108K],    -   [L10V, V18I, D87S, D88S, L101F, K104R, K105T]    -   [R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T]    -   [L10V, V18I, D87S, T88S, L101F, K104R, K105T]    -   [L10V, K89R, L101N, K105E, R108T]    -   [R11S, I14M, T88S, L101N, K105A, R108K]    -   [L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, R108K]    -   [L10V/I; D87S; T88S; K89R; L101H/F; K104R; K105T]    -   [L10T/D; R11I; V18I; R86K; D87K/G; T88S; K89R; L101Y; K104R;        K105T; R108K]    -   [L10A/V; R86T; D87G; T88K; K89R; L101N; K104R; K105A/E; R108K/T]    -   [L10I; V18I; R86T; D87G; T88S; K89R; L101Y/H; K104R; K105A]    -   [L10V; V18I; D87S; T88S; L101F; K104R; K105T]    -   [V18I, R86T, D87G, T88S, L101Y, K104R, K105A]    -   [R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M]    -   [L10V, K89R, L101N, K105E, R108T].        which substitutions are optionally combined with the        substitutions [E12A/G/S, R65D/E].

Polypeptide of the present invention can be further modified, e.g.,joined to a wide variety of other oligopeptides or proteins for avariety of purposes. For example, post-translationally modified, forexample by prenylation, acetylation, amidation, carboxylation,glycosylation, pegylation, etc. Such modifications can also includemodifications of glycosylation, e.g. those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; e.g. by exposing thepolypeptide to enzymes which affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes.

The ability of a molecule to bind to an IL-13 receptor can bedetermined, for example, by binding to an IL-13 receptor coated on anassay plate, displayed on a microbial cell surface, in solution, etc.The binding activity of IL-13 variants of the present invention to areceptor can be assayed by immobilizing the receptor or the IL-13variant to a bead, substrate, cell, etc. Agents can be added in anappropriate buffer and the binding partners incubated for a period oftime at a given temperature. After washes to remove unbound material,the bound protein can be released with, for example, SDS, buffers with ahigh pH, and the like and analyzed.

In some embodiments, a IL-13 variant of the present invention is afusion protein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some other embodiments, the secondpolypeptide is part or whole of Fc region. In some other embodiments,the second polypeptide is any suitable polypeptide that is substantiallysimilar to Fc, e.g., providing increased size and/or additional bindingor interaction with Ig molecules. These fusion proteins can facilitatepurification and show an increased half-life in vivo. Fusion proteinshaving disulfide-linked dimeric structures (due to the IgG) can also bemore efficient in binding and neutralizing other molecules than themonomeric secreted protein or protein fragment alone.

In yet some other embodiments, the second polypeptide is a markersequence, such as a peptide which facilitates purification of the fusedpolypeptide. For example, the marker amino acid sequence can be ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. Wilson et al., Cell 37: 767, 1984. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

In some other embodiments, IL-13 variants of the present inventioninclude IL-13 variants further modified to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. For example, variantsof the present invention further include analogs containing residuesother than naturally occurring L-amino acids, e.g. D-amino acids ornon-naturally occurring synthetic amino acids. D-amino acids may besubstituted for some or all of the amino acid residues.

IL-13 variants of the present invention can be produced by any suitablemeans known or later discovered in the field, e.g., produced fromeukaryotic or prokaryotic cells, synthesized in vitro, etc. Where theprotein is produced by prokaryotic cells, it may be further processed byunfolding, e.g. heat denaturation, DTT reduction, etc. and may befurther refolded, using methods known in the art.

The polypeptides may be prepared by cell-free translation systems, orsynthetic in vitro synthesis, using conventional methods as known in theart. Various commercial synthetic apparatuses are available, forexample, automated synthesizers by Applied Biosystems, Inc., FosterCity, Calif., Beckman, etc. By using synthesizers, naturally occurringamino acids may be substituted with unnatural amino acids. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized. One of skill in the art can readily utilize well-knowncodon usage tables and synthetic methods to provide a suitable codingsequence for any of the polypeptides of the invention. The nucleic acidsmay be isolated and obtained in substantial purity. Usually, the nucleicacids, either as DNA or RNA, will be obtained substantially free ofother naturally-occurring nucleic acid sequences, generally being atleast about 50%, usually at least about 90% pure and are typically“recombinant,” e.g., flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome. The nucleicacids of the invention can be provided as a linear molecule or within acircular molecule, and can be provided within autonomously replicatingmolecules (vectors) or within molecules without replication sequences.Expression of the nucleic acids can be regulated by their own or byother regulatory sequences known in the art. The nucleic acids of theinvention can be introduced into suitable host cells using a variety oftechniques available in the art.

According to the present invention, IL-13 variants can be provided inpharmaceutical compositions suitable for therapeutic use, e.g. for humantreatment. In some embodiments, pharmaceutical compositions of thepresent invention include one or more therapeutic entities of thepresent invention or pharmaceutically acceptable salts, esters orsolvates thereof. In some other embodiments, pharmaceutical compositionsof the present invention include one or more therapeutic entities of thepresent invention in combination with another therapeutic agent, e.g.,another anti-tumor agent.

Therapeutic entities of the present invention are often administered aspharmaceutical compositions comprising an active therapeutic agent and aother pharmaceutically acceptable excipient. The preferred form dependson the intended mode of administration and therapeutic application. Thecompositions can also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

In still some other embodiments, pharmaceutical compositions of thepresent invention can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

Methods of Use

According to yet another aspect of the invention, it provides methodsfor treating, reducing or preventing disease in an individual, includingwithout limitation atopic diseases, by inhibiting the biologicalactivity of IL-13, thereby decreasing IL-13 signaling in the individual.Such methods include administering to a subject in need of treatment atherapeutically effective amount or an effective dose of a high affinityIL-13 polypeptide. Effective doses of the therapeutic entity of thepresent invention, e.g. for the treatment of disease, vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human, butnonhuman mammals may also be treated. Treatment dosages need to betitrated to optimize safety and efficacy.

An “IL-13 associated disorder” is one in which IL-13 contributes to apathology or symptom of the disorder. Accordingly, an IL-13 bindingagent, e.g., an IL-13 binding agent that is an antagonist of one or moreIL-13 associated activities, can be used to treat or prevent thedisorder.

Disorders that can be treated or alleviated by the agents describedherein include those respiratory disorders and asthma caused byinfectious agents, such as viruses (e.g., cold and flu viruses,respiratory syncytial virus (RSV), paramyxovirus, rhinovirus andinfluenza viruses. RSV, rhinovirus and influenza virus infections arecommon in children, and are one leading cause of respiratory tractillnesses in infants and young children. Children with viralbronchiolitis can develop chronic wheezing and asthma, which can betreated using the methods described herein. Also included are the asthmaconditions which may be brought about in some asthmatics by exerciseand/or cold air. The methods are useful for asthmas associated withsmoke exposure (e.g., cigarette-induced and industrial smoke), as wellas industrial and occupational exposures, such as smoke, ozone, noxiousgases, sulfur dioxide, nitrous oxide, fumes, including isocyanates, frompaint, plastics, polyurethanes, varnishes, etc., wood, plant or otherorganic dusts, etc. The methods are also useful for asthmatic incidentsassociated with food additives, preservatives or pharmacological agents.Also included are methods for treating, inhibiting or alleviating thetypes of asthma referred to as silent asthma or cough variant asthma.

The methods disclosed herein are also useful for treatment andalleviation of asthma associated with gastroesophageal reflux (GERD),which can stimulate bronchoconstriction. GERD, along with retainedbodily secretions, suppressed cough, and exposure to allergens andirritants in the bedroom can contribute to asthmatic conditions and havebeen collectively referred to as nighttime asthma or nocturnal asthma.In methods of treatment, inhibition or alleviation of asthma associatedwith GERD, a pharmaceutically effective amount of the IL-13 polypeptidecan be used as described herein in combination with a pharmaceuticallyeffective amount of an agent for treating GERD. These agents include,but are not limited to, proton pump inhibiting agents like PROTONIX®brand of delayed-release pantoprazole sodium tablets, PRILOSEC® brandomeprazole delayed release capsules, ACIPHEX® brand rebeprazole sodiumdelayed release tablets or PREVACID® brand delayed release lansoprazolecapsules.

IL-13 polypeptides can also be useful in treating inflammation andfibrosis, e.g., fibrosis of the liver. IL-13 production has beencorrelated with the progression of liver inflammation (e.g., viralhepatitis) toward cirrhosis, and possibly, hepatocellular carcinoma (deLalla et al. (2004) J. Immunol. 173:1417-1425). Fibrosis occurs, e.g.,when normal tissue is replaced by scar tissue, often followinginflammation. Hepatitis B and hepatitis C viruses both cause a fibroticreaction in the liver, which can progress to cirrhosis. Cirrhosis, inturn, can evolve into severe complications such as liver failure orhepatocellular carcinoma. Blocking IL-13 activity using the IL-13polypeptides described herein can reduce inflammation and fibrosis,e.g., the inflammation, fibrosis, and cirrhosis associated with liverdiseases, especially hepatitis B and C. For example, an IL-13polypeptide can be administered in an amount effective to treat orprevent the disorder or to ameliorate at least one symptom of theinflammatory and/or fibrotic disorder.

In another aspect, this application provides compositions, e.g.,pharmaceutical compositions, that include a pharmaceutically acceptablecarrier and at least one IL-13 polypeptide of the invention.

In another aspect, this application features a method of modulating,e.g., interfering with (e.g., inhibiting, blocking or otherwisereducing), an interaction, e.g., binding, between IL-13 and a cognateIL-13 binding protein, e.g., an IL-13 receptor complex, e.g., a complexcomprising IL-13Rα1 and IL-4Rα, or a subunit thereof. The modulating canbe effected in vivo or in vitro. In other embodiments, the IL-13polypeptide of the invention and interferes with (e.g., inhibits, blocksor otherwise reduces) an interaction, e.g., binding, between IL-13 and asubunit of the IL-13 receptor complex, e.g., IL-13Rα1 or IL-4Rα,individually. In yet another embodiment, the IL-13 polypeptide of theinvention interferes with (e.g., inhibits, blocks or otherwise reduces)an interaction, e.g., binding, between IL-13 and IL-13Rα1. In anotherembodiment, the IL-13 polypeptide of the invention interferes with(e.g., inhibits, blocks or otherwise reduces) an interaction, e.g.,binding, between IL-13 and IL-13Rα1.

The subject methods can be used on cells in vitro (e.g., in a cell-freesystem), in culture, e.g. in vitro or ex vivo. For example, IL-13receptor-expressing cells can be cultured in vitro in culture medium andthe contacting step can be effected by adding an IL-13 polypeptide tothe culture medium. Alternatively, the method can be performed on cellspresent in a subject, e.g., as part of an in vivo (e.g., therapeutic orprophylactic) protocol. For example, the IL-13 polypeptide can bedelivered locally or systemically.

The method can include contacting IL-13 with the IL-13 receptor complex,or subunit thereof, under conditions that allow an interaction betweenIL-13 and the IL-13 receptor complex, or subunit thereof, to occur tothereby form an IL-13/IL-13 receptor mixture.

In some embodiments, the dosage may range from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once every two weeks or once a month or once every 3 to 6months. Therapeutic entities of the present invention are usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, therapeutic entities of the present inventioncan be administered as a sustained release formulation, in which caseless frequent administration is required. Dosage and frequency varydepending on the half-life of the polypeptide in the patient.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor growth, tumor metastasis or tumorinvasion of diseases including lymphomas, leukemias, carcinomas,melanomas, glioblastomas, sarcomas, myelomas, etc. For prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk ofdisease in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease.

Examples of additional therapeutic agents that can be coadministeredand/or coformulated with an IL-13 polypeptide include: inhaled steroids;beta-agonists, e.g., short-acting or long-acting beta-agonists;antagonists of leukotrienes or leukotriene receptors; combination drugssuch as ADVAIR®; IgE inhibitors, e.g., anti-IgE antibodies (e.g.,XOLAIR®); phosphodiesterase inhibitors (e.g., PDE4 inhibitors);xanthines; anticholinergic drugs; mast cell-stabilizing agents such ascromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; andantihistamines. Such combinations can be used to treat asthma and otherrespiratory disorders. Additional examples of therapeutic agents thatcan be coadministered and/or coformulated with an IL-13 binding agentinclude one or more of: TNF antagonists (e.g., a soluble fragment of aTNF receptor, e.g., p55 or p75 human TNF receptor or derivativesthereof, e.g., 75 kd TNFR-IgG (75 kD TNF receptor-IgG fusion protein,ENBREL™)); TNF enzyme antagonists, e.g., TNFα converting enzyme (TACE)inhibitors; muscarinic receptor antagonists; TGF-β antagonists;interferon gamma; perfenidone; chemotherapeutic agents, e.g.,methotrexate, leflunomide, or a sirolimus (rapamycin) or an analogthereof, e.g., CCI-779; COX2 and cPLA2 inhibitors; NSAIDs;immunomodulators; p38 inhibitors, TPL-2, Mk-2 and NFκB inhibitors, amongothers.

The IL-13 polypeptides of the invention may be used in vitro in bindingassays in which they can be utilized in liquid phase or bound to a solidphase carrier. In addition, the polypeptides in these immunoassays canbe detectably labeled in various ways. Examples of types of assays whichcan utilize high affinity IL-13 polypeptides of the invention are flowcytometry, e.g. FACS, MACS, histochemistry, competitive andnon-competitive immunoassays in either a direct or indirect format; andthe like. Detection of IL-13 receptors using the IL-13 polypeptides ofthe invention can be done with assays which are run in either theforward, reverse, or simultaneous modes, including histochemical assayson physiological samples. Those of skill in the art will know, or canreadily discern, other assay formats without undue experimentation.

The IL-13 polypeptides can be bound to many different carriers and usedto detect the presence of IL-13 receptor expressing cells. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingproteins, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbio-luminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the polypeptides of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to thepolypeptides of the invention can be done using standard techniquescommon to those of ordinary skill in the art.

IL-13 receptors may be detected by the IL-13 polypeptides of theinvention when present in biological fluids and tissues. A sample can bea liquid such as urine, saliva, cerebrospinal fluid, blood, serum andthe like, or a solid or semi-solid such as tissues, feces, and the like,or, alternatively, a solid tissue such as those commonly used inhistological diagnosis, for example in tumor tissues.

Another labeling technique which may result in greater sensitivityconsists of coupling the polypeptides to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

Compositions for the treatment of disease can be administered byaerosol, parenteral, topical, intravenous, intratumoral, oral,subcutaneous, intraarterial, intracranial, intraperitoneal, intranasalor intramuscular means. A typical route of administration is intravenousor intratumoral, although other routes can be equally effective.

A composition that includes an IL-13 polypeptide of the invention, canbe formulated for inhalation or other mode of pulmonary delivery.Accordingly, the IL-13 polypeptide can be administered by inhalation topulmonary tissue. The term “pulmonary tissue” as used herein refers toany tissue of the respiratory tract and includes both the upper andlower respiratory tract, except where otherwise indicated. An IL-13polypeptide can be administered in combination with one or more of theexisting modalities for treating pulmonary diseases.

In one example the IL-13 polypeptide is formulated for a nebulizer. Inone embodiment, the IL-13 polypeptide can be stored in a lyophilizedform (e.g., at room temperature) and reconstituted in solution prior toinhalation. It is also possible to formulate the IL-13 polypeptide forinhalation using a medical device, e.g., an inhaler. See, e.g., U.S.Pat. No. 6,102,035 (a powder inhaler) and U.S. Pat. No. 6,012,454 (a drypowder inhaler). The inhaler can include separate compartments for theIL-13 polypeptide at a pH suitable for storage and another compartmentfor a neutralizing buffer and a mechanism for combining the IL-13polypeptide with a neutralizing buffer immediately prior to atomization.In one embodiment, the inhaler is a metered dose inhaler.

The three common systems used to deliver drugs locally to the pulmonaryair passages include dry powder inhalers (DPIs), metered dose inhalers(MDIs) and nebulizers. MDIs, the most popular method of inhalationadministration, may be used to deliver medicaments in a solubilized formor as a dispersion. Typically MDIs comprise a Freon or other relativelyhigh vapor pressure propellant that forces aerosolized medication intothe respiratory tract upon activation of the device. Unlike MDIs, DP'sgenerally rely entirely on the inspiratory efforts of the patient tointroduce a medicament in a dry powder form to the lungs. Nebulizersform a medicament aerosol to be inhaled by imparting energy to a liquidsolution. Direct pulmonary delivery of drugs during liquid ventilationor pulmonary lavage using a fluorochemical medium has also beenexplored.

For example, for administration by inhalation, an IL-13 polypeptide isdelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant or a nebulizer. The IL-13polypeptide may be in the form of a dry particle or as a liquid.Particles that include the IL-13 polypeptide can be prepared, e.g., byspray drying, by drying an aqueous solution of the IL-13 polypeptidewith a charge neutralizing agent and then creating particles from thedried powder or by drying an aqueous solution in an organic modifier andthen creating particles from the dried powder.

The IL-13 polypeptide may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges for use in an inhaler or insufflator may beformulated containing a powder mix of an IL-13 polypeptide and asuitable powder base such as lactose or starch, if the particle is aformulated particle. In addition to the formulated or unformulatedcompound, other materials such as 100% DPPC or other surfactants can bemixed with the IL-13 polypeptide to promote the delivery and dispersionof formulated or unformulated compound. Methods of preparing dryparticles are described, for example, in WO 02/32406.

An IL-13 binding agent, e.g., an anti-IL-13 molecule, can be formulatedfor aerosol delivery, e.g., as dry aerosol particles, such that whenadministered it can be rapidly absorbed and can produce a rapid local orsystemic therapeutic result. Administration can be tailored to providedetectable activity within 2 minutes, 5 minutes, 1 hour, or 3 hours ofadministration. In some embodiments, the peak activity can be achievedeven more quickly, e.g., within one half hour or even within tenminutes. An IL-13 binding agent, e.g., an anti-IL-13 molecule, can beformulated for longer biological half-life (e.g., by association with apolymer such as PEG) for use as an alternative to other modes ofadministration, e.g., such that the IL-13 binding agent enterscirculation from the lung and is distributed to other organs or to aparticular target organ.

Alternatively, compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles such as polylactide, polyglycolide, orcopolymer for enhanced adjuvant effect, as discussed above. Langer,Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of this invention can be administered in theform of a depot injection or implant preparation which can be formulatedin such a manner as to permit a sustained or pulsatile release of theactive ingredient. The pharmaceutical compositions are generallyformulated as sterile, substantially isotonic and in full compliancewith all Good Manufacturing Practice (GMP) regulations of the U.S. Foodand Drug Administration.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition.

Also within the scope of the invention are kits comprising thecompositions of the invention and instructions for use. The kit canfurther contain a least one additional reagent, e.g. a chemotherapeuticdrug, anti-tumor antibody, etc. Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Methods:

Protein expression and purification. Human IL-13 and human IL-13Rα1 andIL-13Rα2-selective variants were cloned into the insect expressionvector pAcGP67 (BD Biosciences) with C-terminal 6×Histidine tag andproduced in insect Hi5 cells using recombinant baculovirus. Proteinswere recovered from Hi5 supernatant after 60 hr of infection by nickelagarose and concentrated and purified by size exclusion chromatographyon a Superdex-200 column into HBS (10 mM Hepes pH 7.4, 150 mM NaCl).biotinylated IL-13Rα1 (amino acids 1-310) and IL-4Rα1 (amino acids1-202) ectodomains were obtained by cloning into the pAcGP67-A vectorwith a C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE andhexahistidine tag. Receptor proteins were coexpressed with BirA ligasewith excess biotin (100 μM).

Surface Plasmon Resonance. SPR experiments were conducted on a BiacoreT100 instrument. Experiments used a Biacore SA sensor chip (GEHealthcare). Biotinylated IL-13Rα1 and IL-13Rα2 receptors were capturedat a low density (100-200 RU) and kinetic runs were conducted at 40μL/min. An unrelated biotinylated protein was immobilized as a referencesurface for the SA sensor chip with matching RU to the experimentalsurface. All data was analyzed using the Biacore T100 evaluationsoftware version 2.0 with a 1:1 Langmuir binding model. Serial dilutionsof unbiotinylated IL-13 variants in the running buffer [1×HBS-P (GEHealthcare)+0.5% BSA] were flowed over the chip and IL-13Rα1/IL-13Rα2were regenerated by using one 60 second injections of 7 mM glycine (pH3.0).

Phospho-flow cytometry assay. The IL-13 responsive cell line A549 wasstimulated with the indicated doses of IL-13 and IL-13 specific variantsfor 15 min. Samples were then fixed in PFA for 15 min at roomtemperature, washed with PBS 0.5% BSA and permeabilized with cold (4°C.) methanol for 10 min. The levels of phosphorylated Stat6 weredetected using a maybe anti-pY641 Stat6 coupled to the fluorophore Alexi488 (BD Bioscience). Analysis was performed on a Becton Dickinson LSRIIequipped with 405, 488, and 640 nm lasers. Data analysis was performedin Citibank software. Log median fluorescence intensity values wereplotted against cytokine concentration to yield dose-response curves.

TF-1 cells proliferation assay. TF-1 cells were seed to 2×10⁵ cells/mlin the presence of the indicated doses of IL-13 or the different IL-13variants for 96 hr. Cells were washed 3× with cold (4C) PBS and fixedwith 4% PFA for 15 min at room temperature. Number of cells in each wellwas determined by flow cytometry. Number of cells were represented aspercentage and plotted against cytokine concentration to obtaindose-response curves.

Dendritic cells differentiation assay. CD14⁺ monocytes were isolated(>97% purity) from peripheral blood mononuclear cells by magneticseparation with anti-CD14 conjugated microbeads (Miltenyi Biotec). 5×10⁵CD14⁺ monocytes were subsequently cultured with 50 ng/mL GM-CSF alone orwith the indicated concentrations of IL-13 in the presence of 2 μg/ml ofisotype control anti body, anti IL-4Rα1 anti body or IL-13dn in 2 mlwell plates containing IMDM medium (Gibco) supplemented with 10% humanAB serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine,sodium pyruvate, non-essential amino acids and 50 μM 2-ME. Cells wereprocessed on day 6 with 5 mM EDTA and subsequently stained with DAPI(Invitrogen), fluorescently labeled isotype control mAbs, or mAbsagainst CD14, CD86, CD209 and HLA-DR (BD Biosciences). Dendritic celldifferentiation was assessed by flow cytometry with a BD LSRII flowcytometer and median fluorescent intensities were generated by FlowJo(Treestar).

In vivo test of IL-13dn efficacy. 360 ng of mouse IL-13 were injectedintra-tracheally with or without 150 μg of IL-13dn on days 0, 3 and 5.Lungs were harvest on day six. RNA was extracted and the expressionlevels of Muc5ac, Periostin, Arg1, CHIA, YM1, Fizz1 were assessed byquantitative PCR.

What is claimed is:
 1. A polypeptide comprising: a modified interleukin13 (IL-13) engineered to have increased affinity for interleukin 13receptor α2 (IL-13Rα2), relative to native human IL 13 protein; (b) anddecreased affinity for interleukin 13 receptor α1 (IL-13Rα1) relative tonative human IL 13 protein; wherein the modified IL-13 polypeptidecomprises amino acid changes relative to wild type IL-13 at positionsL10, R86, D87, T88 and R108.
 2. The modified IL-13 polypeptide of claim1, comprising a set of amino acid substitutions: [L10H, R86T, D87G,T88R, R108K].
 3. The modified IL-13 polypeptide of claim 2, comprisingan amino acid sequence set forth in SEQ ID NO:18.
 4. The polypeptide ofclaim 1, conjugated or fused to a therapeutic or labeling moiety.
 5. Thepolypeptide of claim 4, wherein the therapeutic moiety is a heterologouspolypeptide fused to the modified IL-13 polypeptide.
 6. The polypeptideof claim 5, wherein the heterologous polypeptide is a cytotoxin.
 7. Thepolypeptide of claim 5, wherein the heterologous polypeptide comprisesan IgG Fc region.
 8. The polypeptide of claim 1 in a pharmaceuticalformulation.
 9. A modified IL-13 antagonist polypeptide engineered tohave decreased affinity for interleukin 13 receptor α2 (IL-13Rα2),relative to native human IL 13 protein; (b) and increased affinity forinterleukin 13 receptor α1 (IL-13Rα1) relative to native human IL 13protein, wherein the modified IL-13 polypeptide comprises amino acidchanges relative to wild type IL-13 at positions V18, D87, T88, L101,K104, and K105; and comprising amino acid substitutions E12A/G/S andR65D/E, wherein the polypeptide is conjugated or fused to a therapeuticor labeling moiety.
 10. The modified IL-13 polypeptide of claim 9,comprising a set of amino acid substitutions selected from [L10V, V18I,D87S, T88S, L101F, K104R, K105T]; [V18I, R86T, D87G, T88S, L101Y, K104R,K105A]; and [R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M].11. The polypeptide of claim 9, wherein the therapeutic moiety is aheterologous polypeptide fused to the modified IL-13 polypeptide. 12.The polypeptide of claim 11, wherein the heterologous polypeptide is acytotoxin.
 13. The polypeptide of claim 11, wherein the heterologouspolypeptide comprises an IgG Fc region.
 14. The polypeptide of claim 9in a pharmaceutical formulation.
 15. A nucleic acid encoding thepolypeptide of claim
 1. 16. The nucleic acid of claim 15, furthercomprising a sequence encoding a heterologous polypeptide fused to themodified IL-13 coding sequence.
 17. The nucleic acid of claim 15,wherein the nucleic acid is present in a vector.
 18. A nucleic acidencoding the polypeptide of claim
 9. 19. The nucleic acid of claim 18,wherein the nucleic acid is present in a vector.